Cocaine induces apoptosis in embryonic mouse neurons : teratogenic implications

The developing central nervous system appears particularly vulnerable to cocaine exposure. However, whether the effects of cocaine on the developing brain are the consequences of prolonged episodes of hypoxia induced by this drug or result from a direct toxicity is still a matter of debate.
The purpose of this work was to investigate direct effects of cocaine on cerebral cells. We have used the embryonic mouse coculture system of neuronal and glial cells, which maintains communication between these two major populations of cerebral cells, to test for direct neurotoxicity of cocaine concentrations that can be expected in the fetal brain upon severe maternal abuse, on developing brain cells, without intervening structures, including placental, maternal and/or fetal systemic effects. In particular, the system of cultures cells eliminates the most important presynaptic sites of cocaine action, namely the re-uptake of neurotransmitters and the metabolic and degradative pathways present in systemic and brain parenchymal compartments. We are aware that the exposure of our coculture system to cocaine does not reflect exactly the “in vivo” pharmacokinetics of fetal exposure. Results from animal studies suggests that cocaine concentrations peak in the fetal brain 20-30 minutes after administering cocaine to the pregnant dam, and fall progressively, with no detectable cocaine after 6 hours (Sandberg et al., 1991, 1995; Spear et al, 1989). In our coculture system, cocaine hydrolyzed with t1/2 of 17.5 hr, involving a different pattern of exposure.
neurons and glial cells were obtained from brain hemispheres of 15-day-old mouse embryos, and were co-cultures for up ti 6 days with Minimum Essential Medium supplemented by heat-inactivated fetal calf serum. After 2 days, and daily thereafter, freshly dissolved cocaine was added to the culture at various concentrations, and medium was renewed daily. Once such cultures were established (after 2 days), neurons lay and extended neuritis on a flat layer of glial cells.
The effects of cocaine were first examined by immuno-fluorescence. After 6 days of coculture, control preparations showed neurons with extensive neuritis and prominent MAP2 labeling, well spread over a flat layer of glial cell, labelled for GFAP. After 4 days of exposure to 100µM cocaine, neuritis became scantly and less branched. At 250µM, the number of neuritis par cells had largely decreased, producing an aspect of bi-and unipolar cells. At 500µM, neuritis had almost completely vanished and very few scattered MAP2-labaled round cell bodies persisted. In contrast the number of glial cells remained unchanged in control and treated cultures, although the drug caused some cell flattening and a discrete alteration in their fibrillar structure. When cells were exposed only for 2 days to cocaine and were then cultured for 2 additional days on cocaine-free medium, the remaining cells reextended neuritis although a definitive loss of cells is evident